Impact of Ramadan fasting on serum levels of major endocrinology hormonal and biochemical parameters in healthy non-athlete adults: A systematic review and meta-analyses

Background Ramadan Intermittent Fasting (RIF) has the potential to alter hormonal levels in the body. This study investigates the impact of RIF on hormonal levels among healthy individuals during Ramadan. Methods A systematic review and meta-analysis of previously published studies were conducted, focusing on healthy non-athlete adults. The intervention examined was Ramadan Intermittent Fasting, and the primary outcomes assessed were changes in endocrine hormonal and biochemical parameters. The pooled effect measure was expressed as odds ratio (OR) and 95% confidence interval (CI) using the random-effects model. Results A total of 35 original articles were retrieved, with a combined sample size of 1,107 participants eligible for the meta-analysis. No significant relationship was found between pre- and post-Ramadan hormonal levels of T3, T4, TSH, FT3, FT4, Testosterone, LH, FSH, Prolactin, PTH, Calcium, and Phosphorus (P-value<0.05). However, a substantial decrease in morning cortisol levels was observed across the studies (P-value: 0.08, Hedges’ g = -2.14, 95% CI: -4.54, 0.27). Conclusions Ramadan Intermittent Fasting results in minimal hormonal changes and is a safe practice for healthy individuals. The fasting regimen appears to disrupt the circadian rhythm, leading to a decrease in morning cortisol levels.


Introduction
Ramadan, the ninth month of the Islamic lunar calendar, holds significant religious importance for the global Muslim community.One of the Five Pillars of Islam, Ramadan, mandates a month-long period of fasting from dawn to dusk for healthy adult Muslims [1,2].This period of intermittent fasting, which extends roughly between 11 to 18 hours per day, is a unique metabolic state that has sparked considerable scientific interest over the past few decades [3].
Given the shift in eating patterns, energy consumption, and sleep cycles, Ramadan fasting can influence various physiological and biological processes, potentially affecting the body's hormonal and biochemical landscape [4][5][6].Various hormones such as insulin, cortisol, leptin, and ghrelin, which are critical regulators of our metabolism, might undergo changes during this fasting period.Similarly, the fasting state can also impact key biochemical parameters, including glucose, lipid, and protein metabolism [7,8].
Several individual studies have looked into these effects, with some suggesting significant changes, while others report minimal or no impacts.The rationale for our systematic review rests on its broader analytical lens, examining both major endocrine hormones and key biochemical parameters.While we acknowledge and have cited existing reviews that focus on specific aspects such as glucometric markers [9], our work aims to offer a more comprehensive assessment of the metabolic and hormonal changes associated with Ramadan fasting in healthy non-athlete adults.In line with the PICOS framework, our research objective is: (P) Healthy non-athlete adults; (I) Observing Ramadan fasting; (C) Measurements before the onset of Ramadan (pre-fasting values); (O) Changes in serum levels of T3, T4, TSH, FT3, FT4, Testosterone, LH, FSH, Prolactin, PTH, Calcium, Phosphorus, and morning cortisol; (S) Observational studies, case-control studies, and quasi-controlled studies.Through this review, we aim to discern whether and to what extent Ramadan fasting influences these critical endocrine parameters in the specified population.

Ethical approval
The study protocol was reviewed and accepted by the ethics committee of the Endocrinology and Metabolism Research Institute of Tehran University of Medical Sciences (ID: IR.TUMS.EMRI.REC.1395.00143).

Registration
The protocol of this systematic review was registered a priori in PROSPERO (CRD42020135123).

Database searches
This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines [10].

Search strategy
A comprehensive literature search was carried out on the Web of Science, PubMed, Scopus, Cochrane Library, and Embase databases in September 2023.The search focused on full published papers in any language.Given the nature of Ramadan fasting, studies evaluated hormonal and biochemical changes before and after Ramadan; therefore, only observational studies were included.Our search strategy combined keywords such as: "Fasting", "Ramadan", and "Hormones" along with their Medical Subject Headings (MeSH) terms.All retrieved papers were imported into Endnote Reference Manager.Duplicates were identified and excluded using the "remove duplicates" function and manual checks.Additional articles were sourced by hand-searching reference lists of included articles and using Google Scholar.

Study selection
Three reviewers (SHM and MP and JSH) independently screened the titles and abstracts of identified studies and full-text reports of eligible studies were obtained for data extraction.Where there was a lack of consensus an expert consultation was performed.

Exclusion criteria
Studies on special populations like athletes, pregnant or lactating women, and patients with different illnesses.Studies using specimens other than serum for parameter evaluations (e.g., serum vs. salivary for cortisol).
Studies providing results solely through graphs without numerical data.Conference abstracts, case reports, editorials, review articles, letters, animal studies, and unpublished papers (since they did not undergo peer-review process).
Studies that are published in predatory journals according to Bell's list Inaccessibility of the full text even after contacting the corresponding author thrice via email.

Grouping for syntheses
By Parameter of Interest: Studies will further be grouped based on the specific hormone or biochemical parameter they are analyzing.

Main outcomes and measures
Main outcome measures consisted of the mean changes in serum levels of parameters of interest in terms of before Ramadan, during Ramadan (if applicable), end of Ramadan, and post Ramadan (if applicable): TSH, T4, T3, LH, FSH, testosterone, cortisol, prolactin, PTH, calcium, and phosphorous.Among the included studies these parameters were the main frequently used outcome that were available.

Data extraction
Data extraction was performed by two mentioned investigators.Information extracted from articles was first author name, year of published study, design, participants' characteristics (sample size, gender, and age, fasting time and place, duration of fasting (hours) and time measures of hormones.

Quality assessment of studies
The JBI (Joanna Briggs Institute) Critical Appraisal Checklist was used by three reviewers independently to appraise the methodological quality of the included studies [11,12].Bias was appraised through nine questions that aim to evaluate the research design of the study followed by the similarities of participants in any comparison and the type of care they received checklists consist of items to assess selection, the existence of a control group, the existence of multiple measurements, follow-up description, the existence of same outcome measurement, reliability of outcome measurements, and the appropriate statistical analysis used.Each criterion had four possible responses including "Yes," "No," "Unclear," or "not applicable.JBI checklist is not a scoring system for grading the studies as well as lack of any consensus about cut-off point for defining the inclusion of the study [13].(Table 1)

Strategy for data synthesis
Qualitative literature review.The evidence of changes in hormonal levels and biochemical parameters during Ramadan Fasting were narratively synthesized, separately.However, the conclusion from the narrative review was planned to be validated by conducting a subsequent meta-analysis.
Quantitative literature review (meta-analysis).Continuous variables were presented as means and standard deviations (SD).Whenever an article reported standard error (SE) instead of standard deviation, standard deviations were calculated based on the formula association among them (SE = SD/ p n). Categorical (binary) data were reported as n (%).whenever it was possible to conduct a meta-analysis, it was performed with STATA software, version 17(Stata Corp, USA).
Heterogeneity Assessment: The Chi-square-based Q-test was used to assessing heterogeneity and was considered to be statistically significant at P < 0.1.The degree of heterogeneity was estimated using I 2 statistic.In overall, the I 2 value of 25% was considered low, the I 2 value of 50% was considered as moderate, and the I 2 value of 75% and more was considered as high heterogeneity [14].Due to the limited number of included studies in each group, a deeper exploration of the sources of heterogeneity was not feasible.Specifically, methodologies such as subgroup analysis, meta-regression, and sensitivity analyses were not conducted due to the insufficient quantity of studies, making these analyses less meaningful and potentially misleading.
Effect Size Measurement: The Fixed-effect model was performed, when no significant statistical heterogeneity between studies were detected (P>0.2), a in case of presenting any heterogeneity random-effects model was used (p�0.2).Hedges' g value was used for effect size measurement and also forest plots were used to present the results graphically and to illustrate point estimates of the effect size and 95% confidence interval (CI).An effect size of �0.2 was described as a small effect, an effect size around 0.5 as a medium effect, and an effect size around 0.8 was as a large effect [15].If Hedges' g CI did not contain 0, In addition to Hedges' g values and the mean difference's forest plot is illustrated.Publication Bias: Funnel plots and Egger's test were applied to detect potential publication biases.

Characteristics of studies
Through searches across multiple databases and other resources, we identified 13,085 papers relevant to our search strategy.After eliminating duplicate documents, we assessed the titles and abstracts of 8,826 publications, which culminated in the full-text retrieval of 96 papers.Eventually, we included 35 publications (encompassing 1,107 participants: 783 men, 271 women, and 53 unknown gender) in our final analysis (Fig 1) [1,5,7,.The included studies spanned between 1982 and 2021.Detailed information quality assessment of studies can be found in Table 1.Notably, no studies were removed due to quality assessment.
Among the selected studies, nineteen focused solely on men, four exclusively on women, while twelve reported results for both genders (Table 2).Two studies did not specify the participant number by gender.The mean age of included participants was 27.99 ± 6.17 years, ranging from 17-56.All the studies implemented a pre-post design to report changes in hormones and/or biochemical markers.Notably, none of the studies included a control group of nonfasted participants.("+" = "yes," "−" = "no," "N/A" = not applicable and "?" = "unclear") https://doi.org/10.1371/journal.pone.0299695.t001Interestingly, one study investigated the effects of Ramadan fasting on two different regions in Jordan [32].The mean fasting hours reported across 28 studies ranged from 11 to 17 hours (13.44±1.97hours).
We categorized the effects of Ramadan fasting according to the hormonal and biochemical markers studied: Thyroid hormones, Cortisol, Sex hormones, and Calcium, Phosphorous, and Parathyroid hormone (PTH).

Effect of Ramadan on cortisol
Three studies evaluated changes in cortisol levels during 24-hours before and after Ramadan fasting.They all found a diurnal decrease in cortisol levels and nocturnal increase in serum cortisol levels during Ramadan fasting [27,48,49].
Two studies evaluated cortisol in the morning and in the evening [28,40].Dwivedi et al. [28] reported a significant reduction in morning cortisol levels, and significant rise in evening cortisol levels compared to pre-Ramadan values in 12 young healthy Muslims men.However, Bahijri et al. [40] did not observe any significant change in morning cortisol values (p = 0.06), but significant rise in evening cortisol values (p = 0.01) two weeks in to Ramadan in 23 young Ramadan practitioners compared to pre-Ramadan cortisol values.Four studies evaluated the impact of Ramadan fasting on morning cortisol levels [23,28,40,46].Analysis of pooled data of participants showed a large insignificant decrease (P = 0.08) in morning cortisol levels as it is shown in Fig 3.

Effect of Ramadan fasting on sex hormones
In overall 8 studies assessed the changes of Sex hormones (Testosterone, LH, FSH) among men during Ramadan fasting.Analysis of pooled data of participants did not show a significant change in Testosterone [1,17,20,30,32,36,42,44], LH [1,17,20,32,36,44], FSH [1,17,20,36,44], Prolactin [17,20]  El-Migdadi et al. [32] assessed serum LH and testosterone values pre-and post-Ramadan fasting in two groups healthy male individuals from two different regions of Jordan country.They found that fasting caused a significant increase in serum LH and testosterone values only in subjects of in the below sea level environment.
One study by Shahabi et al. evaluated the effect of Ramadan fasting on LH, FSH levels and Ovulation in females; and concluded that Islamic fasting causes neither significant variation in the secretion of hormones around ovulation nor does it influence the occurrence of ovulation [37].

Discussion
The objective of our systematic review and meta-analysis was to elucidate the impact of Ramadan fasting on the serum levels of major endocrinology hormones and biochemical parameters in healthy non-athlete adults.Our analysis incorporated data from a diverse array of studies, capturing variations in geographical location, gender, age, and fasting duration.
We found no significant changes in thyroid hormones (TSH, T3, T4), sex hormones (Testosterone, LH, FSH, Prolactin), and key biochemical markers (Calcium, Phosphorous, PTH) during Ramadan fasting across genders.However, we did observe a trend towards diurnal decrease and nocturnal increase in serum cortisol levels.While these findings were not statistically significant, they may still suggest physiological adaptations to the altered eating and sleeping patterns during Ramadan.
Our findings are in alignment with the notion that intermittent fasting during Ramadan does not induce drastic changes in endocrine functions or biochemical parameters in healthy individuals.This could potentially be attributed to the adaptive capacity of the human body, which has evolved mechanisms to maintain metabolic and hormonal homeostasis even during periods of fasting.
Ramadan fasting represents a well-recognized model of religious intermittent fasting that significantly alters eating and sleep patterns for an entire month.These changes could potentially disrupt the normal functioning of various body organs.However, several researchers have suggested that intermittent fasting may indeed have beneficial effects on human health [50,51].Numerous original research studies, systematic reviews, and meta-analyses have been conducted to evaluate the effects of Ramadan fasting on various health-related factors in both healthy and non-healthy individuals.It is important to note that as the Islamic world spans across diverse geographical locations, featuring different dietary patterns and social habits, the fasting effects observed in Ramadan practitioners from various regions are likely to differ [27].
During Ramadan, alterations in the sleep-wakefulness cycle and changes in social habits might potentially affect the circadian pattern of certain hormonal parameters.These include pituitary hormones (PRL, LH, FSH, GH, and TSH), steroid hormones (cortisol, testosterone), and thyroid hormones [52].Our study found that Ramadan Intermittent Fasting could result in a considerable yet statistically insignificant decrease in Morning Cortisol levels.Furthermore, most of the reviewed studies reported a significant rise in Evening Cortisol levels.The consensus from these studies was that these changes were largely due to alterations in circadian rhythm during Ramadan.
The findings regarding changes in thyroid hormones during Ramadan were somewhat mixed.According to the results of included studies, Ramadan fasting does not appear to adversely affect thyroid function.Any observed changes in thyroid-related hormones remained within normal limits during this month.

Effect of Ramadan fasting on glucometabolic markers
Our study initially intended to conduct a meta-analysis on glucometabolic markers.However, during our review process, an exhaustive systematic review by Faris et al. was published [9].In light of their recent work, we instead performed a systematic update for newer studies on glucometabolic markers.Three new studies observed a minor rise in blood sugar levels (one of which was statistically insignificant) [53][54][55].Additionally, one study reported a slight and insignificant rise in serum insulin levels [53].None of these findings contradicted the previously mentioned systematic review.
Our meta-analysis revealed no significant changes in TSH, total T4, and total T3 during Ramadan.A significant reduction was, however, observed regarding free T4 values.In women, total T4 and T3 hormone concentrations may decrease due to alterations in binding proteins in the last days of Ramadan, but free hormone indices remain within normal limits [5,24].

Limitations
It is crucial to highlight the high heterogeneity observed for several parameters, which could be due to methodological differences across studies, such as the timing of sample collection, the participant's age, gender, and lifestyle factors.This heterogeneity underscores the need for standardized protocols in future studies to ensure comparability and consistency of findings.Despite the lack of a control group of non-fasted participants in the included studies, our review still offers valuable insights into the physiological adaptations during Ramadan fasting.It should be noted, though, that our findings may not apply to specific populations such as athletes, elderly, or those with underlying health conditions, and therefore caution must be exercised when generalizing the findings.

Conclusion
This meta-analysis findings suggest that Ramadan fasting has not any adverse effect on glucometabolic markers, sexual hormones, thyroid hormones, calcium, phosphorus, and PTH.There was a near significant decrease of serum morning cortisol level, which it is due to the change in circadian rhythm.Ramadan Intermittent Fasting imposes minimum hormonal change, and is a safe practice.
Population (P): Healthy adults without limitations regarding gender, ethnicity, and geographical area.Intervention (I): Observing the effects of Ramadan fasting on the parameters of interest.Comparison (C): Changes in the parameters of interest before (baseline-a few days before or the first day of Ramadan) and after the completion of Ramadan or at least 2 weeks into the fasting month.Outcome (O): Levels of hormones/biochemical parameters of interest.Study design (S): Observational studies published in peer-reviewed journal of any language.